Abstract Methionine sulfoxide reductases (MsrA and MsrB) are repair enzymes that reduce methionine sulfoxide (MetO) residues in proteins to methionine (Met) in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. Mammals possess one MsrA and three MsrBs. The major mammalian MsrB, MsrB1, is a selenocysteine-containing protein, whose expression and activity can be regulated by dietary selenium. In this application, we propose to characterize regulation of protein function by stereospecific, site-specific, reversible Met oxidation; define the roles of Met, MetO and enzymes that act upon them in the regulation of lifespan; and establish a systems-level understanding of Met oxidation. Specific Aim 1 is based on the finding that mouse Mical proteins specifically oxidize conserved Met residues in actin, which are then reduced back to Met by MsrB1. Further studies may add MetO to a list of posttranslational modifications, such as phosphorylation, acetylation, and methylation, that regulate protein function. We will characterize Mical-based oxidation of Met residues in actin and MsrB-based reduction of MetO residues, determine the redox state of Met in actin using unique reagents and examine physiological relevance of actin regulation through reversible Met oxidation. Specific Aim 2 will address discrepancy in the regulation of lifespan by various Msrs. We will use transgenic fruit flies expressing various Msrs to test the hypothesis that the reduction of free MetO contributes to lifespan control by these enzymes. In addition, we will employ Met and MetO diets to test the hypothesis that the regulation of lifespan by Met depends on the status of other amino acids. Specific Aim 3 will identify proteins with MetO by using advanced proteomic methods and characterize Met reactivity on the proteome scale. We will also employ stereospecific MetO sensors to characterize these species in cells and biological samples.